Development of loop-mediated isothermal amplification (LAMP) assay for detection of Hepatozoon canis infection in dogs.

The laboratory diagnosis of canine hepatozoonosis, caused by Hepatozoon canis is tedious, especially in chronic and latent infections. In the present investigation, a loop mediated isothermal amplification (LAMP) assay was developed and standardized targeting the partial 18S rRNA gene (GenBank accession no. KU096058). The LAMP primers specifically amplified H. canis DNA, whereas no amplification was detected in DNA samples from dogs infected with Babesia vogeli, B. gibsoni, Ehrlichia canis and Trypanosoma evansi, and no amplification was observed in DNA samples from H. canis-free dogs. The threshold sensitivity level of the assay was determined to be 15 fg of genomic DNA of H. canis. Furthermore, evaluation of blood samples collected from 250 dogs presented at Small Animal Clinics, Teaching Veterinary Clinical Complex, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab (India) was carried out for the presence of H. canis by microscopy, 18S PCR assay and LAMP assay. Of the total samples subjected to these tests, LAMP detected H. canis in 75 samples, while 18S PCR and microscopy detected H. canis in 28 and 9 samples, respectively. The present investigation has developed, for the first time, a highly sensitive, specific and rapid LAMP assay for the detection of H. canis, which has practical applications for the screening of field samples.